分子信标用于p53 mRNA的体外定量检测  

Quantitative Detection of P53 mRNA by Molecular Beacons in vitro

作　　者：何丽芳[1] 唐红星[1] 王柯敏[1] 谭蔚泓[1] 刘斌[2] 孟祥贤[2] 李军[1] 王炜[1] 

机构地区：[1]湖南大学化学化工学院化学生物传感与计量学国家重点实验室,长沙410082 [2]湖南大学生物医学工程中心

出　　处：《化学学报》2006年第11期1116-1120,共5页Acta Chimica Sinica

基　　金：国家重点基础研究发展规划(No.2002CB513110);科技部国际合作重点项目(No.2003DF000039);国家科技攻关计划项目(No.2003BA310A16);国家自然科学基金(No.20305006)资助项目.

摘　　要：根据p53基因的序列设计并合成了能特异性检测p53mRNA的分子信标(MB),发展了一种快速定量测定细胞内总RNA提取物中p53mRNA的方法.采用鼻咽癌(CNE2)细胞系和经RNA干扰技术降低p53基因表达的CNE2-p53RNAi细胞系,抽提总RNA并用MB检测,验证了MB的检测对象是p53mRNA.将该方法应用于多种肿瘤细胞内p53基因表达水平的分析,表达变化趋势与经典的mRNA分析方法RT-PCR检测结果相符.在此基础上,用MB对5-氟尿嘧啶(5-Fu)处理的肺腺癌细胞(A549)进行了p53mRNA的体外定量检测,结果表明采用MB能够快速地获知该药物对细胞内p53mRNA表达影响的信息.A p53 molecular beacon （p53 MB） designed according to the p53 gene sequence was synthesized to quantitatively detect p53 mRNA in vitro. The specificity of the p53 MB was tested by hybridizing the MB with total RNA extracted from CNE2, CNE2-vector （CNE2 cells transfected with empty plasmid） and CNE2-p53 RNA interference （CNE2 cells transfected with p53 RNA interference plasmid） cell lines. Lower fluorescence intensity in CNE2-p53 RNA interference cell samples confirmed the specificity of the p53 MB to the p53 mRNA. The change of the p53 mRNA expression in various kinds of cell lines was measured by traditional RT- Polymerase Chain Reaction （PCR）. Based on this, the p53 mRNA expression in the lung carcinoma cells of A549 treated with 5-fluorouracil at various time was detected by p53 MB in vitro. These results indicated the rapid response of p53 MB to the effect of the drug on the expression of p53 mRNA in cells.

关 键 词：分子信标 P53 RNA干扰 5-氟尿嘧啶 定量 

分 类 号：Q522.2-33[生物学—生物化学]